Electronics zap DNA into pores to read its sequence

Researchers use some custom electronics to turn DNA copying on and off by …

The second generation of DNA sequencing machines have now taken over the market, and their high throughput is producing new genomes at a staggering clip. But the third generation of machines are already in the development pipeline, with features like longer DNA reads, faster speeds, and single-molecule precision. Over the weekend, Nature Nanotechnology published a paper on one of the promising technologies that's currently in the works: nanopore sequencing. It reports some preliminary success in developing a computerized nanopore system that controls when DNA bases are added, and reads them one-by-one in the process.

The term "nanopore" would seem to imply a bit of carefully structured metal; it's anything but. The pore in question is simply a protein that embeds in a membrane and creates a tiny passage through it, just big enough to fit a single strand of DNA. Since DNA carries a negative charge, applying a voltage across the membrane can drive a single strand of DNA through the pore (the double helix won't fit); as it travels through, small voltage changes result that can be used to "read" the sequence. The problem is that the molecules tend to fly through too fast for a clear signal to be picked up, so various tricks are being considered in order to slow things down, like chewing up a DNA molecule one base at a time.

The new paper suggests that it might be possible to use the same voltage differences used to send the DNA through the pore to control how quickly bases are added to DNA by an enzyme called polymerase, which normally copies the molecule (we've described DNA polymerases in detail previously). The process starts by getting a polymerase to stick to a strand of DNA, ready to make a copy of it. When a voltage is applied, the DNA moves through the pore, but the polymerase is too big, and gets stuck on the outer surface.

When it's jammed up against the surface of the pore, the polymerase can't add any bases; the whole system is stuck. That's where electronics come in. The authors set up a field programmable gate array to run a Finite State Machine that steps the system through a cycle. The first step is to lower the voltage difference, which lets the DNA snake backwards through the pore. This frees the polymerase, which takes a few milliseconds to add a base. The voltage is then reapplied, and the system senses whether a new base has been added, creating a longer stretch of double helix and "pulling" the single stranded section back out of the pore, one base at a time. With a new base stuck in the pore, it should be possible to read it.

Right now, the system has a lot of rough edges: it misses some bases, only works for short stretches of DNA, and doesn't discriminate between bases very well. But the authors claim that there are ways that all of these issues could be improved. And, even if it doesn't beat some of the alternatives to market, it's a very interesting mix of biochemistry and electronics.

7 Reader Comments

Nanopore sequencing like this, or Oxford Nanopore's approach (glue an exonuclease to the pore, feed your DNA strand to the exonuclease which cleaves the bases to fall into the pore) are the most exciting of the next gen sequencing technologies IMO. Sadly they also seem the most far off.

"And, even if it doesn't beat some of the alternatives to market, it's a very interesting mix of biochemistry and electronics"

A good example of what is likely to be an emerging trend. That is hybrid technologies that mix electronics, engineered biochemical systems, and possibly other forms of nanotechnology. It probably will not happen in the next few years. But it probably is essentially already in the pipe for sometime this century.

I work on a detection scheme using electrodynamic redox facilitated by DNA strands, so I am familar with ths work. The main problem in my estimation is that creating microarrays is very expensive with this technique.

"Right now, the system has a lot of rough edges: it misses some bases, only works for short stretches of DNA, and doesn't discriminate between bases very well. But the authors claim that there are ways that all of these issues could be improved." Which is exactly what everyone says about their technique. I guess I'd encourage people not to hold their breath.

They are using α-Haemolysin as their pore. The problem with it is that it has a long narrow constriction so that approximately 15 bases contribute to the current level.

Since they are not reading while the polymerase is working, the polymerase could be adding any number of bases, making reading repetitive regions problematic. Also, polymerases tend to fall off quite frequently: they do not capture DNA like a pore, but grab on like a hand.

Speed is not the only problem. Even with an immobilized homo-polymer, Adenine and Guanine, both purines, are not well resolved. Also, the larger size of purines tends to mask the presence of pyrimidines within the pore.

Due to the stochastic nature of nanoscale physics, long integration times are necessary. Combining this with the poor resolution of bases means that without a major improvement in the pore, sequencing with a nanopore will take seconds per base. While nanopore sequencing is the most promising technique for indefinite read lengths, I doubt it will be competitive with Sanger derived techniques within 10 years, if ever.

Wow... it IS my field, and I barely kept up with what you said (granted, I do a l lot more physics than bio in my biophysics). I'm impressed that you know so much in something that is not your field, hats off.

But what you said is exactly right, dynamic DNA sequencing produces all kinds of problems like this, which is why traditional sequencing is done with immobilized DNA references (which take the place of the pores here) and then allowing the DNA to hybridize. Not that that is devoid of its own set of problems.

Still, these guys have a very discrete signal in their paper, which is what we all want (and what most of us don't get), so if they are particularly clever with how they go about this, they might be onto something.